This invention relates to myeloma cell lines adapted for growth and cloned in a serum-free medium in which transferrin is the sole protein supplement. Such cell lines may be used in the construction of hybridomas by fusion with immunocytes, e.g. splenocytes.
The development of somatic-cell hybrids as introduced by Kohler and Milstein in 1975, allows for the production of monoclonal antibodies with a desired specificity. These antibodies are functionally homogeneous, which greatly facilitates the standardization of immunochemical procedures and assures reproducibility in established assay systems. It is often necessary to obtain these antibodies in a highly purified state. One approach is to cultivate the antibody secreting hybrid cells in a chemically defined medium; that is, one free from any type of serum or serum supplement.
Serum is a very complex fluid, containing at least 500 different protein components with a total protein concentration approaching 150 mg/ml. Hybridoma culture medium is routinely supplemented with 5%-20% serum. This introduces a tremendous excess of heterologous protein beyond monoclonal antibody protein concentration, typically 1-100 mg/ml. The advantage of producing monoclonal antibodies in a serum-free system is evident strictly from a purification standpoint. Serum components may, however, interfere with assays for antibody reactivity, and, in any event, add considerably to the cost of the medium.
In attempting to develop serum-free cell lines, others have liberally added a variety of protein supplements, such as insulin, to minimal growth media. These supplemented media partake to some degree of the disadvantages of serum media because their protein components may be difficult to obtain free of contaminants, and to separate from the desired antibody product.
Rossi, et al., Am J. Vet. RES., 41:1680-1681 (1980); Chu, et al., In Vitro, 9:31-34 (1973); and Nuttall, et al., Nature, 266:835-837 (1977) all address the problem of viral contamination of serum. These viruses may interact biologically with mammalian cells in culture, as noted by Chu, or obscure clinical reactions to other agents, as observed by Nuttall.
A nubmer of techniques have been employed to alleviate viral contamination. These include regular screening of cell cultures, and removal of the viruses by affinity chromatography. Unfortunately, as reported by Orr, et al., J. Clin. Microbiol., 3:402-5 (1976), it is difficult to assure the elimination of all the different types of undesirable agents that may be present in a contaminated culture.
In contrast, we have found transferrin to be a well characterized and easily purified protein.
Work relating to the present invention was described in Abstract No. 123, Brown, Shriver, Harshman and Rener, Adaptation of a Murine Myeloma Cell Line to Serum-Free Media and Its Use as a Fusion Partner in Monoclonal Antibody Production, In Vitro (March 1984). The abstract, while mentioning use of transferring, does not state it was the sole protein source.
Kawamoto, et al., Hybridoma Formation in Serum-Free Medium in Hormonally Defined Media: Tool Cell (Biol (1983) 1 Meet. 310-13, abstracted in Derwent Biotechnology Abstracts, 84-06338 (July 2, 1984), describes a multipart basal medium (RPMI, Ham's F-12, and DME, combined) with many supplements (insulin, human transferrin, 2-aminoethanol, 2-mercaptoethanol, sodium selenite, human low density lipoprotein, oleic acid, and fatty acid-free bovine serum albumin) utilized for the development of NS-1 myeloma-derived hybridomas in serum-free media. The medium of the present invention utilizes a single basal medium (IMDM) and a single protein supplement (human transferrin). This sole protein source is a small, well characterized, and easily purified molecule.
Golde, U.S. Pat. No. 4,438,032 describes a human T-lymphoblast cell line (Mo) that, while preferentially grown in a 20% FCS medium, is said to be capable of growth, at a considerably slower rate, in serum-free medium. Nor does he teach use of this cell line as a fusion partner in construction hybridomas.
Galfre, U.S. Pat. No. 4,350,683 describes rat myeloma cell line CNCM I-078, said to be capable of growth in serum free IMDM. He teaches that his cell line may be useful in the development of rat-rat hybridomas. There is no reference to transferring as a sole protein source.
Lundak, EP Appl. No. 62,409 describes a lymphoblastoid cell line capable of growth in serum-free Iscove's medium. This cell line is promoted as a fusion partner for use in hybridoma construction. However, the hybridomas were constructed in a serum-containing medium. Additionally, the parent cell line was grown in a medium which, although serum-free, was conditioned with insulin. Similarly, Chang et al., J. Immunol. Meth. 39:369-75 (1980), describe growth of hybridoma cell lines in serum-free media containing insulin in addition to transferrin.
Transferrins are iron-binding vertebrate proteins. Their nomenclature is rather inconsistent, as noted by Bezkorovainy and Zschocke, Arzheim-Forsch. (Drug Res.) 24:476-485 (No. 4, 1974), and references to "transferrins" are intended to encompass all iron-binding proteins of the transferring class, however derived, unless a specific transferring is clearly indicated.
Tormey, et al., Exper. Cell Res., 74:163-169 (1972) identified transferrin, a constituent of human serum, as a lymphocyte growth promoter. Earlier, Vogt, et al., Exper. Cell Res., 54:195-200 (1969) had reported that the transferring in fetal bovine serum potentiated DNA synthesis in cultured mouse spleen cells.
Hayashi and Sato taught, Nature, 359:132-134 (1976), that the primary role of serum in cell culture is to provide hormones, and that an established rat pituitary cell line could be grown in a serum free medium supplemented with thyrotropin-releasing hormone, transferrin, the biologically active peptide of parathyroid hormone, nd a partially purified somatomedin preparation.
Guilbert and Iscove first found that serum could be partially replaced by Selenite, transferrin, albumin and lecithin in hematopoietic cell cultures. Nature, 263:594-595 (1976). Later, they achieved complete replacement of serum by albumin, transferring and soybean lipid in cultures of liposaccharide-reactive B lymphocytes. In their view, the role of serum is confined "mainly and perhaps entirely to supplying transferrin, lipid and albumin to the cells." J. Exper. Med., 147:923-933 (1978).
Ventrex, in a 1984 circular (SL 001 1/84) on HL-1, "Completely Defined Serum Free Media," states that "for myeloma to grow in HL-1, Supplemental Fetal Bovine Sera . . . must be added." A 0.5% to 1.0% FBS supplement is recommended for P3x63/Ag8.653 derivatives. HL-1 itself contains insulin in addition to transferrin.
Other references of interest are Cartaya, U.S. Pat. No. Re. 30,985, and Moldenhauer, U.S. Pat. No. 4,282,326.